Crystallins comprise the protein-rich tissue of the eye lens. Of the three most common vertebrate subtypes, β-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the β-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized β-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl2 or MgCl2). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate β-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge-properties that can be reversed when salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms of β-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.

This study aims to identify the underlying genetic defects of β-crystallin (CRYB) genes responsible for congenital cataracts in a group of Chinese families.
Detailed family history and clinical data of six Chinese families with autosomal dominant congenital cataracts were recorded. Targeted exome sequencing was applied to detect the underlying genetic defects for the families. Generated variants were confirmed by PCR and sanger sequencing. Afterward, bioinformatic analysis through several computational predictive programs was performed to assess impacts of mutations on protein structure and function.
A total of 53 participants (23 affected and 30 unaffected) from six unrelated Chinese families were recruited. Cataract phenotypes covered nuclear, total, posterior polar, pulverulent, snowflake-like, and zonular. Through targeted exome sequencing, six mutations in four β-crystallin genes were revealed which included five missense mutations CRYBB1 p.Q70P, CRYBB2 p.E23Q, CRYBB2 p.A49V, CRYBB2 R188C, CRYBA4 p.M14K and one splice mutation CRYBB3 c.75+1 G>A. In silico results predicted pathogenic for all four missense variants except variant CRYBB2-p.A49V yielded results as tolerant. The CRYBB3 c.75+1 G>A splice site mutation was predicted to be deleterious by leading to a broken splice site, a premature stop codon, and subsequently resulting in a short peptide of 113 amino acids, which may affect protein features.
The obtained results expanded mutational and phenotype spectrum of β-crystallin genes and offer clues for pathogenesis of congenital cataracts. The data also demonstrated that targeted exome sequencing is valuable for providing molecular diagnostic information for congenital cataract patients.

The crystallins (α, β and γ), major constituent proteins of eye lens fiber cells play their critical role in maintaining the transparency and refractive index of the lens. Under different stress factors and with aging, β- and γ-crystallins start to unfold partially leading to their aggregation. Protein aggregation in lens basically enhances light scattering and causes the vision problem, commonly known as cataract. α-crystallin as a molecular chaperone forms complexes with its substrates (β- and γ-crystallins) to prevent such aggregation. In this chapter, the structural features of β- and γ-crystallins have been discussed. Detailed structural information linked with the high stability of γC-, γD- and γS-crystallins have been incorporated. The nature of homologous and heterologous interactions among crystallins has been deciphered, which are involved in their molecular association and complex formation.

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.

Ion mobility mass spectrometry was employed to study the structure of the βB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of βB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on βB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased βB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of βA3/βB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of βB1 and βA3/βB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among βB1 solubility, stability, aggregatory propensity and function of protecting βA3.